Device and method for investigating analytes in liquid suspension or solution

ABSTRACT

An optical detection device is provided for analysing analytes in a liquid suspension or solution that can detect and process a large number of wavelengths of incident and fluorescent light simultaneously, which is small in size and can be easily adapted to different investigation requirements. In one embodiment an optical detection device comprises a light supplying means ( 45 ), an analyte handling means ( 78 ), a light directing means ( 19 ), and detection means, integrated on planar substrate devices ( 40 ), ( 20 ), and ( 30 ), ( 30 ′), respectively. A plurality of optical waveguides are integrated in the substrate devices to direct light emitted by the light supplying means ( 45 ) through the different sections of the optical detection device to the detection means. The analyte handling means ( 78 ) comprises an analyte channel ( 70 ) for the liquid flow of the analyte suspension or solution and an analyte sorting means ( 72 ) comprising several sorting channels ( 72 ′).

This application claims priority to PCT/GB02/05567, filed Dec. 9, 2002,published on Jul. 3, 2003, Publication No. WO 03/054525 A2 in theEnglish language and which claimed priority to GB 0129688.8, filed Dec.12, 2001.

FIELD OF THE INVENTION

The present invention relates to a device and a method for investigatinganalytes in liquid suspension or solution.

BACKGROUND OF THE INVENTION

Flow cytometry is a well-known fluorescent analysis technique, whichcan, for example, be used to detect and separate blood cells in asuspension according to their phenotypic properties. This can beachieved by staining the cells in the sample to be analysed for examplewith a monoclonal antibody specific to a phenotypic marker, which itselfis coupled to a fluorescent dye. Different phenotypic cell surfacemarkers can therefore be distinguished when dyes with differentfluorescent wavelengths, e.g. green and red, are coupled to a number ofdifferent antibodies, each specific to a different marker.

To perform this analysis technique a Fluorescence Activated Cell Sorter,generally called FACS, is known, which is a complex device comprisingoptical and fluid-handling subsystems, which are usually assembled fromdiscrete components. An example of such a FACS is disclosed in Roitt,Brostoff, Male, Immunology 5^(th) edition, Mosby Publishers (1998),p385, and is shown in FIG. 1. In FIG. 1 a fluorescent-stained samplecell suspension 1 is introduced into a vibrating flow cell 2.

A cell flow 4 passing out of the flow cell 2 is encased in a sheath ofbuffer fluid 3, which has been separately introduced into the vibratingflow cell 2. The flow cell uses the principle of laminar flow andhydrodynamic focussing. A laminar flow of liquid, i.e. a flow that isnon-turbulent, passing through a cylindrical tube will be subject to aviscous drag at the wall of the tube that leads to a higher flowvelocity near the tube centre. The resulting velocity profile is that ofa parabola. The so called Bernoulli effect associates such adifferential velocity profile with a pressure gradient that pointsradially inwards from the tube wall, i.e. the pressure in the liquidflow decreases from the wall to the centre of the tube. This pressuregradient will move any particle in suspension in the flow into thecentre of the flow where it will remain. To prevent blocking, asuspension of particles can be introduced into the tube through a widebore which is surrounded concentrically by a larger bore containingsheath fluid. By constricting this coaxial flow whilst maintaininglaminar flow a focused stream of particles can be obtained. If asuspension of discrete particles is introduced into the tube in thismanner the particles will flow through the centre of the tube insequence, aligned behind one another. The sheath fluid commonly used inflow cytometry is phosphate buffered saline solution or a similarelectrolyte solution which can be charged electrically.

The flow 4 passing out of the flow cell 2 is illuminated by a laser 5within an interrogation region. The emitted laser beam is scattered atthe fluorescent-stained sample cells in the suspension in acharacteristic manner according to the specific optical properties ofthe particles or analytes in the suspension and the fluorescent dyesused, to detect specific markers.

Scattered and fluorescent light passing from the cell flow and emergingfrom the interrogation region is collected by a light directingsubsystem with a plurality of beam splitters 6, collimating andfocussing lenses (not shown) and is directed to a detection subsystemwith several detectors 7, 8, 9, 10. Detector 7 measures forward scatterof incident light from the laser, which allows for an estimation of thesize of the cells in the flow 4 passing the laser 5. Similarly thegranularity of cells can be detected, by collecting light scattered at a90 degree angle with detector 8 after the light has been redirected byone of the beam splitters 6. Finally, detectors 9 and 10 detect, forexample, red and green fluorescence, emitted by green and redfluorescent dyes, respectively, to identify surface markers present onthe cells. This is achieved by collecting fluorescent light emitted inthe same 90 degree path leading away from the flow 4 and by passing thelight through a second beam splitter in the plurality of beam splitters6 to illuminate the detectors 9 and 10. The detectors 9 and 10 aretherefore designed to only detect light emitted in the green and redwavelength bands of interest, respectively.

Where possible, fluorescent dyes will be used that have a commonexcitation wavelength maximum that can be excited by a single lightsource and different emission wavelength maxima, such as in the green orred spectrum. Most commercial flow cytometry systems in use today willdetect between 2 and 6 fluorescent wavelengths. The detectors themselvesare in most cases photomultiplier tubes and the wavelengthdiscrimination is normally achieved by inserting bandpass filters in thelight path before the entry facet of the receiving tube. Because of thesize of the individual components in the detection subsystem it ischallenging to manage the spatial arrangement of all optical componentswhen a system with a relatively large number of detectors is required.In such a situation, the lengths of different light paths from the flowcell to the respective detectors may vary substantially betweendifferent detectors. These constraints tend to limit the number offluorescent wavelengths that are typically used in commercialapplications today.

For the event that cell sorting may be required, the end of the flowcell 2 has a vibrating nozzle, which causes the flow 4 emanating fromthe flow cell into air to break up into droplets that usually contain nomore than a single cell. These droplets, falling from the nozzleperpendicular towards the ground due to their initial velocity andgravity, enter into an analyte sorting subsystem, where they aresubsequently passed through a charging collar 11, which applies asubstantially uniform electrical charge to each droplet. When sorting isrequired, the data received by the detectors 7, 8, 9, 10 can beprocessed to steer electrostatic deflection plates 12 under computercontrol, which allows re-directing different cell populations in theflow 4 at different angles into different ones of a plurality of outputsample tubes 13, according to the fluorescent and other opticalproperties detected.

In an alternative embodiment mechanical sorting is known wherein eitherthe final receptacle collecting the flow emanating from the flow cell orits inlet is switched electromechanically in order to receive therelevant fraction of cells.

As mentioned before, typical commercial FACS devices in use today candistinguish between 2 and 6 fluorescent colours. At the same time theyare bulky and expensive limiting their broad use in research andclinical practice. They require precision bulk optical and discretemechanical components. At the same time, however, the requirement ofresearchers to distinguish a higher number of variables in analytesamples is increasing.

Cell sorters that detect a larger number of colours have been designed,but are not used in wide practice. This is mainly due to the fact thatextending the existing bulk optical and fluidics technologies to highernumbers of colours that can be discriminated is very expensive andcumbersome.

There are also known first attempts of miniaturising FACS-devices. Forexample a conventional flow chamber of a FACS is replaced bymicrofluidic devices manufactured by micromachining technology like softlithography. In a paper “A micorfabricated fluorescence-activated CellSorter” of Anne Y. Fu et al., Nature Biotech., Vol17, p. 1109 (November1999) there is described a FACS for sorting various biological analytes.A cell sorting device is produced as a silicone chip with channels for asample liquid. The chip is mounted on an inverted microscope, andfluorescence is excited near junctions of the channels with a focusedlaser beam generated by a bulk laser, which is directed onto the chipperpendicularly to the plane of the chip. The fluorescent emission iscollected by the microscope and measured with a photomultiplier tube,which collects light emitted perpendicularly to the plane of the chip.The laser beam is focused perpendicular to the chip and a plurality ofbeam splitters, mirrors, etc. is necessary to guide the light.

U.S. Pat. No. 5,726,751 discloses a flow cytometer made of twocomponents: a flow cytometer optical head and a disposable flow module.The flow module utilises a flow channel micromachined in a siliconwafer. The optical head comprises a laser to provide an illuminatingbeam and photodetectors. The laser and the photodetectors are arrangedout of plane of the wafer, dependent on the angle of the analysing lightbeam, so that the photodetectors may collect the analysing beam.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide an optical detectiondevice for analysing analytes in a liquid suspension or solution thatcan be scaled to detect and process a large number of wavelengths ofincident and fluorescent light simultaneously, which is small in sizeand can be easily adapted to different investigation requirements toenlarge the possibilities of its application.

According to a first aspect of the present invention, there is provideda device for analysing analytes in a liquid suspension or solutioncomprising, an analyte handling means including an analyte input region,an analyte channel for carrying analytes in a liquid suspension orsolution comprising a light receiving region for receiving light from alight supplying means to illuminate the analytes in the interrogationregion, an analyte output region, a light guiding means for directinglight emerging from the analytes in an interrogation region of theanalyte channel to an optical detection means for detecting one or moreproperties of the analytes in the suspension or solution, characterisedin that at least the analyte channel and a first optical waveguide forguiding light emerging from the analytes in the analyte channel areintegrated on the same first planar substrate.

Preferably, a second optical waveguide guiding light emerging from theanalytes in the analyte channel is integrated on the first planarsubstrate.

Preferably, a third optical waveguide guiding light incident on theanalytes in the analyte channel is integrated on the first planarsubstrate.

Preferably, the first optical waveguide is interfacing with the analytechannel, such that it collects light travelling in the analyte channelat an angle of between 30 and 60 degrees from the longitudinal axis ofthe analyte channel.

Preferably, the first optical waveguide is interfacing with the analytechannel, such that it collects light travelling in the analyte channelat an angle of substantially 45 degrees from the longitudinal axis ofthe analyte channel.

Preferably, the second optical waveguide is interfacing with the analytechannel, such that it collects light travelling in the analyte channelat an angle of between 60 to 120 degrees from the light collected by thefirst optical waveguide.

Preferably, the second optical waveguide is interfacing with the analytechannel, such that it collects light travelling in the analyte channelat an angle of substantially 90 degrees from the light collected by thefirst optical waveguide.

Preferably, doped absorbing regions may be provided on the firstsubstrate adjacent to the analyte channel and the waveguide(s) forreducing the amount of unguided light propagating in the firstsubstrate.

Preferably the refractive index of any or all of the waveguidessubstantially matches the refractive index of the analyte suspension orsolution. Alternatively, or in addition, a single or multiple dielectriccoating may be formed at the interface between the waveguides(s) and theanalyte channel.

Preferably, the waveguide(s) may be tapered at their interface with theanalyte channel.

According to another aspect of the invention, the third waveguidedivides into two waveguides for injecting light into two spatiallyseparate interrogation regions in the analyte channel and at least onewaveguide is provided for collecting light emerging from the analytesadjacent to each one of the separate interrogation regions.

According to yet another aspect of the invention there is provided amethod for investigating analytes in a liquid suspension or solution,particularly by using an optical detection device as described above.

According to yet another aspect of the present invention an analytesorting means is provided for sorting different analytes with respect totheir properties, which may have been detected by the optical detectiondevice. The analyte sorting means includes an analyte channel with ananalyte input for introducing the liquid suspension or solution of theanalytes and a plurality of sorting channels comprising at least oney-junction in a staged cascade that is integrated on a substrate,wherein opposite polarity electrodes are formed in the substrate oneither side of the at least one y-junction each for one investigatedoptical property of the analytes.

An analyte sorting means designed in this way enables the separation ofanalytes even of very small sample amounts.

In still another aspect of the invention, there is provided a lightsupplying means for use with a device for analysing analytes in a liquidsuspension or solution, wherein one or several components of the lightsupplying means are integrated on a second planar substrate.

Preferably the light supplying means comprises at least one or morelight emitting diodes or laser diodes are attached to the second planarsubstrate.

Preferably the light supplying means comprises at least one integratedoptical waveguide for carrying light from the light emitting diode(s) orlaserdiode(s) is leading from the light emitting diode(s) orlaserdiode(s).

Preferably the light supplying means comprises several waveguides areleading from each one in an array of light emitting diodes orlaserdiodes and a dispersive element is integrated on the second planarsubstrate for combining light of different wave-lengths λ₁ . . . λ_(m)received from the waveguides into a single output waveguide.

Preferably the dispersive element is an arrayed waveguide grating or atransmission grating formed by an array of recesses etched into thesecond substrate.

In still another aspect of the present invention, there is provided alight directing means for use with a device for analysing analytes in aliquid suspension or solution, wherein at least one dispersive element,at least one optical waveguide for carrying light towards the dispersiveelement, and at least one plurality of output waveguides collectinglight of wavelengths or wavelength bands λ₁ . . . λ_(m), λ′₁ . . .λ′_(n) to be separated by the at least one dispersive element areintegrated on a third planar substrate.

Preferably the dispersive element(s) used in any of the light supplyingand light directing means is/are (an) arrayed waveguide grating(s) or atransmission grating formed by an array of recesses etched into thesecond substrate.

Preferably, one dispersive element, one optical waveguide for carryinglight towards the dispersive element, and one plurality of outputwaveguides collecting light of wavelengths or wavelength bands λ₁ . . .λ_(m), λ′₁ . . . λ′_(n) to be separated are provided each for separatinglight received from a forward scatter path and for separating light froma side scatter path leading from an analyte interrogation region.

Preferably any of the first, second and third substrate is made of anyof silica and silica-on-silicon and silicon-on-insulator.

In still another aspect of the present invention, there is provided adetection means for use with a device for analysing analytes in a liquidsuspension or solution, wherein at least one optical detector isintegrated in or hybridised on a fifth planar substrate.

Preferably, a plurality of optical detectors is provided with onedetector each being provided for detecting a different one of thewavelengths λ₁ . . . λ_(m), λ′₁ . . . λ′_(n).

Preferably the detectors are photodiodes.

Preferably, each detector is mounted on top of one of several v-groovesterminating in an inclined end face formed in the fifth substrate thev-grooves extending to the edge of the substrate.

Preferably, the fifth substrate is butt-coupled to the third substrateand bonded by means of an appropriate glue or resin.

Preferably, each in the plurality of detectors receives light from adifferent waveguide in at least one of the pluralities of outputwaveguides integrated in the third planar substrate.

In another aspect of the invention, an array of detectors is providedeach for receiving light transmitted from a forward scatter path andlight transmitted from a side scatter path leading from an analyteinterrogation region.

In another aspect the invention provides for a device for analysinganalytes in a liquid suspension or solution, including an analytehandling means, an analyte sorting means, a light supplying means, alight directing means, and the light detecting means as describedherein.

In another preferred embodiment of the present invention, at least twoof the analyte sorting means, the light supplying means, the lightdirecting means, and the light detecting means are connected by opticalfibres. Releasable connections between optical fibres leading from therespective means may be made by means of optical fibre connectors.

In yet another preferred embodiment of the present invention, at leasttwo of the analyte sorting means, the light supplying means, the lightdirecting means, and the light detecting means are integrated on thesame planar substrate.

Another aspect of the invention concerns the use of the an opticaldetection device as described herein for investigatomg the properties ofone or more analytes in a liquid suspension or solution.

Preferably, use of an optical detection device as described herein ismade, wherein the optical properties of the one or more analytes in thesuspension or solution are marked by markers with known opticalproperties and the one or more wavelength or wavelength band(s) λ₁, . .. , λ_(m) of the light supplying means are selected according toemission and absorbtion maxima of the used markers.

Preferably, the markers for the analytes are fluorescent.

Preferably, sorting of the analytes of the suspension or solutionaccording their different optical properties is acheived by guiding theminto different sorting channels according to the different propertiesdetected.

In another aspect, the invention concerns a method for investigatinganalytes in a liquid suspension or solution using an optical detectiondevice as described herein.

Preferably the method for investigating analytes in a liquid suspensionor solution using an optical detection device as described herein iscarried out, wherein at least two wavelenghts or wavelength bands λ₁, .. . , λ_(n), λ′₁, . . . , λ′_(n) in the light emerging from teh analytesare detected simultaneously.

The flow of the analyte suspension or solution through the analytechannel and the sorting channels may be reversible.

Manufacturing each of the different means of the optical detectiondevice on separate semiconductor devices and connecting these devices byoptical fibres, etc., substantial distances can be bridged. Therefore itis e.g. possible to arrange the analyte handling means in anexperimental hood, use light supplying means, that is installed in aseparate room, and set up the detection means in still a further room.

By designing a device for analysing analytes in a liquid suspension orsolution according to the present invention a very compact and highchannel count system can be realised cost-effectively. This allowssaving valuable bench-space in laboratory or point-of-care applications,or even the realisation of a hand-held device. The optical detectiondevice can be manufactured, to a large extent with high-volumesemiconductor processing technologies like lithographic patterning,masking and etch processes or the like, which allow for the realisationof very high specification and narrow tolerance devices in high volumesand at low cost. The possibility of arranging the different sections ofthe optical detection device and the discrete components of thesesections independently from one another on the substrates permits anassembly of the device, that can be optimised according to certainrequirements, e.g. of the analytes, the light source or the components.The present invention will therefore enable use of a describedflow-cytometric and similar industrial and/or biomedical analytedetection and handling applications in new areas of medical andscientific use.

By integrating the channel and the waveguides on the surface of a planarsubstrate the total amount of space required by the optical detectiondevice can be reduced substantially.

Through using the components and processes described herein knowncomponents and techniques the device for analysing analytes can bemanufactured very cost efficiently. Additional objects and advantages ofthe invention will be set forth in the detailed description whichfollows, and will be obvious from the description. Preferred or optionalfeatures of the invention will also be apparent from the appendedclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the invention may be more readily understood embodimentswill now be described, merely by way of example, with reference to theaccompanying drawings in which:

FIG. 1 is a schematic view of a known fluorescence activated cell sorteras known in the prior art;

FIG. 2 a is a schematic top view of an optical detection deviceaccording to one embodiment of the present invention;

FIG. 2 b is a schematic top view of an optical detection deviceaccording to another embodiment of the present invention;

FIG. 2 c is a schematic perspective view of a part of the opticaldetection device of FIG. 2 b showing a substrate covering an area withinthe analyte handling means;

FIG. 3 a is a schematic enlarged top view of the detection means of theof the optical detection device of the present invention;

FIG. 3 b is a schematic enlarged top view of an alternative embodimentof the waveguide interfaces with the analyte channel in the opticaldetection device;

FIG. 3 c is a schematic enlarged top view of the layout of the liquidchannels and optical waveguides within the analyte handling means;

FIG. 4 a is a cross section along line A—A in FIG. 3 c, showing theanalyte channel and buried waveguides in the optical detection device;

FIG. 4 b is a cross section along line B—B in FIG. 3 c, showing theanalyte channel and buried sorting elements;

FIG. 4 c is a cross section along line C—C in FIG. 3 c, showing theanalyte input of the analyte handling means of the present invention;

FIG. 5 a is a cross section view, showing the analyte input regioncomprising an additional system for hydrodynamic focussing;

FIG. 5 b is a schematic view of the external fluid handling arrangementof an analyte handling means;

FIG. 6 is a schematic representation of the optical output spectrum ofthe optical detection device of the present invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

In FIG. 2 a an embodiment of an optical detection device according tothe present invention is shown, which comprises a light supplying means45 integrated on a planar substrate device 40, an analyte handling means78 integrated on a planar substrate device 20, a light directing means19 integrated on a planar substrate device 80, and detection meansintegrated on planar substrate devices 30, 30′. Also shown is aplurality of optical waveguides, which are integrated in the substratedevices and are designed to direct light emitted by the light supplyingmeans 45 through the different sections of the optical detection deviceto the detection means. In the analyte handling means 78 there is shownan analyte channel 70 for the liquid flow of the analyte suspension orsolution and an analyte sorting means 72 comprising several sortingchannels 72′ for different properties of the analytes.

The planar substrate devices 20, 40, 80 may for example be a silica orsilica on silicon substrate and optical waveguides formed in thesubstrates may be buried channel waveguides formed by methods well knownin the art of integrated optics, such as described in WO0125827.Alternatively the substrates 20, 40, 80 may be silicon-on-insulator andthe waveguides formed as silicon-on-insulator ridge waveguides methodsfor forming integrated silicon on insulator ridge waveguides aredescribed in WO9508787 and in a paper entitled “Low Loss Single ModeOptical Waveguides with Large Cross-Section in Silicon on Insulator” byJ. Schmidtchen et al in Electronic Letters, 27, p.1486, 1991. Furthermethods for manufacturing silicon-on-insulator optical waveguides aredescribed in WO0036445, WO0025156. The planar substrate device 30 may beformed may be silicon.

The light supplying means 45 may be a discrete wavelengthmultiwavelength light source for emitting light with a predeterminedcharacteristic, which is changeable by interaction of the light withanalytes in suspension or solution according to the properties of theanalytes. If the system is used for detecting fluorescence, a m-channeldevice may have a light source emitting m discrete wavelengths or narrowwavelength bands λ₁, . . . , λ_(m) which are selected according to theproperties of the analytes to be investigated. For example, λ₁, . . . ,λ_(m) may be within a narrow tolerance of the absorption maxima of nselected fluorescent markers to be used in detection, which can beexcited to fluoresce on fluorescent wavelengths or narrow wavelengthbands λ′₁, . . . , λ′_(n). The fluorescent markers used may be knownfluorescent chemical or biological molecules or fluorescent beads orparticles that have characteristic fluorescent properties due to theirchemical properties and or physical dimensions. Preferably none of thewavelengths or wavelength bands λ₁, . . . , λ_(m), λ′₁, . . . , λ′_(n)overlap to an extent where it would be difficult to distinguish thesignals carried in them. In practice the overlap should be preferablyless than −20 dB and more preferably less than −30 dB. If all absorptionmaxima of the n fluorescers are different in wavelength from one anotherm will be equal to n. However, in case some of these absorption maximaoverlap m will be less than n.

Therefore there are provided m light emitters 44 generating light ofwavelengths or wavelength bands λ₁, . . . , λ_(m). The light emitters 44may be distributed feedback lasers (DFB lasers) or external cavitylasers, as described in WO0003461, that are integrated or hybridised onthe substrate 40 in a linear array. Methods for hybridising an activelight emitting element on a substrate in alignment with an outputwaveguide is described, for example in WO9743676. The active lightemitting element in this assembly is typically formed from a combinationof type II, III, V, VI semiconductor materials according to methods wellknown in the art. If an external cavity laser is chosen and thesubstrate material in which waveguides are formed is silica, then thegrating forming the front reflector of the cavity may be provided byusing methods similar to those well known in the art for forming opticalgratings in telecommunications fibres. A waveguide 43 is leading fromeach of the light emitters 44 to a dispersive element 42 that is used tomultiplex the light output from the various light emitters 44 into asingle waveguide 41, which leads to the edge of the substrate 40 whereit connects to the fibre 50.

If silicon-on-insulator is chosen for the substrate 20, 40, 80, then theoptical analysis will have to be carried out at wavelengths greater than1100 nm, as silicon is not transparent at wavelengths shorter than 1100nm. This does not pose a problem, however, as optical components such aslaser diodes and photodetectors are commonly available for suchwavelength requirements. Fluorescent dyes or particles have to be usedaccordingly, which have both their absorption and emission maxima atwavelengths greater than 1100 nm.

Using a dispersive element 42 in the multiplexing process is well knownin the art and has the advantage of minimising transmission losses.Examples of forming such dispersive elements can be found for example inU.S. Pat. No. 5,029,981, and U.S. Pat. No. 5,467,418, both of whichdescribe arrayed waveguide type elements well known in the art ofintegrated optics. In these cases a plurality of unequal lengthwaveguides (the waveguide array) causes the necessary phase differencein the light front travelling through the array to generate the desireddispersion. Another embodiment of a dispersive element is disclosed inEP 0365125, in which a one-dimensional reflective-type diffractiongrating is formed in an slab waveguide by a one-dimensional array ofwells formed with walls that are substantially perpendicular to theplane of the substrate 40.

The connection between the fibre 50 and the waveguide 41 can be carriedout by means well known in the art, using methods such as described, forexample, in WO 97042534, WO9835250, WO9839677, WO9957591, and WO0129598,and by other methods well known in the art. The fibre may be a multimodeor more preferably a single mode silica fibre, such as commonly used intelecommunications applications. A typical single mode optical fibrewill have a 125 micrometer silica cladding and a 8 micrometer singlemode silica core whereby the silica in the core has a higher refractiveindex than the silica in the cladding.

The fibre 50 connects via a connector 51 to an input fibre 52 to theedge of the planar substrate device 20. The connector 51 may be astandard fibre-to-fibre connector, such as those commonly used formaking connections between optical telecommunications fibres. On thesubstrate 20 an optical waveguide 21 collects the multi-wavelength lightbeam received from the light supplying means 45 in the fibre 52. Theconnection of fibre 52 to waveguide 21 can be provided in the samemanner as described above for the connection between fibre 50 andwaveguide 41.

The waveguide 21 directs the light beam to the analyte channel 70located on the planar substrate device 20. The liquid suspension orsolution of the analytes is introduced into the analyte channel 70 viathe input port 71 and flows through the channel 70 towards an analytesorting means 72 (described in more detail later on), comprising aplurality of sorting channels 72′ that each lead to one in a pluralityof output ports 74. The analyte channel 70, the analyte sorting channelsin the plurality 72′, the input port 71, and the output ports in theplurality 74, are formed in the substrate 20 as an extended pattern ofrecesses that are formed by machining into the top surface of thesubstrate or patterned by standard lithographic processes and etchedinto the top surface of the substrate using processes well known in theart of semiconductor manufacturing, such as reactive ion etching.

Light is injected into an interrogation region 18 within the analytechannel 70 from the waveguide 21. If the waveguides are formed in asilica or a silica-on-silicon substrate, the waveguide 21 interfaceswith the analyte channel 70 at an angle, such that light refractedacross the interface of the analyte channel 70 with the waveguide 21will enter the analyte suspension or solution carried in the channel atan angle of 30 to 60 degrees to the axis of the analyte channel, andmore preferably at an angle of approximately 45 degrees. Analytespresent in an analyte suspension are labelled with fluorescent dyes. Aswill be described in more detail later on the analyte suspension passesthrough the analyte cannel 70, travelling past the interrogation region18 where the analytes are illuminated by the light received from thewaveguide 21. The analytes in the suspension have been pre-stained,according to methods well known in the art of flow cytometry, with arange of n fluorescent dyes each specific to a given phenotypic propertyof the analytes to be detected, illuminating of the stained analytes bylight injected into the interrogation region 18 from the waveguide 21causes a combination of absorption, scattering and fluorescence in theanalytes within the interrogation region 18.

Preferably the refractive index of the waveguide 21 and that of theanalyte suspension or solution in the analyte channel 70 are matched inorder to avoid significant reflection losses. Such index matching can beachieved, for example by varying the buffer formulation for the analytesuspension or by doping the optical waveguides used. In an embodimentwhere a residual mismatch between the analyte suspension or solution andthe optical waveguides on the substrate 20 cannot be avoided,antireflective coatings may be formed on the sidewalls of the analytechannel 70 in the interrogation region 18 according to processes wellknown in the art of integrated optics to reduce backreflections at theinterfaces further. In such an event the angle of refraction at suchinterfaces will also have to be taken into account according to thestandard principles of wave optics, with the aim that light injected inthe analyte suspension will travel at an angle of 30 to 60 degrees tothe axis of the analyte channel 70, and more preferably at an angle ofapproximately 45 degrees. On the opposite side of the analyte channel 70after the light has passed the interrogation region 18 forward-scatteredlight is collected by waveguide 27 and 90 degree scattered andfluorescent light is collected by waveguide 26. Consequently thewaveguides 26 and 27 are arranged to collect light travelling at anangle of 0 and 90 degrees within the analyte suspension or solution,respectively, with respect to the angle of incident light travelling inthe analyte suspension or solution. If there is no index mismatchbetween the suspension or solution and the waveguides, this translatesto an angle between the waveguides 26 and 27 of 90 degrees, but in asituation where these refractive indexes are not matched, differentangles between the waveguides 26 and 27 and between these waveguides andthe side face of the analyte channel will have to be determinedaccording to standard principles of wave optics. Usually the purpose ofdetecting forward scattered light is to determine the size of theanalyte particles and the purpose of detecting 90 degree scattered lightis to detect the granularity of these particles, as well as anyfluorescence emitted by them. A particular advantage of this arrangementis that it is possible to collect fluorescent light at a higher signalto noise ratio than when it is collected in the forward scatter path.The waveguides 21, 26, 27 may conveniently be tapered at the interfacewith the analyte channel 70 to improve the optical transmissioncharacteristics of their interfaces.

The cross section of the waveguides 21, 26, 27 at the interface with theanalyte channel 70 will preferably be in the region of 1 micron to 10micron diameter and more preferably between 3 micron and 8 microndiameter. Preferably, the analyte channel 70 will have a cross sectionbetween 5 micron and 100 micron diameter and more preferably between 10micron and 25 micron diameter.

The light emerging from the interrogation region 18 after interactionwith the analytes in the liquid suspension or solution is guided by thewaveguides 26 and 27 to the edge of the substrate 20 where they connectto optical fibres 53 and 56, respectively. Light transmitted to thesefibres is transmitted via fibre optic connectors 54 and 57 to opticalfibres 55 and 58, respectively, which transmit light guided in thesefibres to the light directing means 19 integrated on the substrate 80.The optical fibres 53, 55, 56, 58 and connectors, 54, 57 are similar tothose components described above for connecting the light supplyingmeans 45 to the analyte handling means 78. Light received from theoptical fibres 55 and 58 is collected by the waveguides 82 and 81. Lighttravelling in the waveguides 81 and 82 subsequently passes to theintegrated dispersive elements 28, 28′. The dispersive elements 28, 28′are designed similarly to the dispersive element 42 on the lightsupplying means 45, preferably either as an arrayed waveguide grating oras a transmission or reflection grating described before. The purpose ofthe dispersive elements 28, 28′ is to divide the light travelling in thewaveguides 81, 82 into a number of discrete wavelength bands to bedetected in the forward and side scatter path from the analyte handlingmeans 78, respectively. Typically, the wavelengths to be detected willbe the wavelengths λ₁, . . . , λ_(m) emitted by the light supplyingmeans 45 and the wavelengths λ₁, . . . , λ_(n) emitted by thefluorescent markers used. Normally m will be less or equal to n, andtherefore the maximum number of wavelength bands to be detected will beless or equal to 2n. The m+n discrete wavelength bands separated by thedispersive elements 28, 28′ are directed towards the pluralities ofwaveguides 29, 29′, respectively, such that each separate wavelengthband will be collected by a different one of the waveguides in thepluralities 29, 29′ as a separate wavefront. Preferably the wavelengthcrosstalk between adjacent waveguides in the pluralities 29, 29′ will bebetter than −20 dB and more preferably better than −30 dB, as shown inFIG. 6. Although the wavelength spectrum shown in FIG. 6 shows equallyspaced wavelength bands, equal spacing of the bands is not required, andthis can be adjusted, according to the fluorescent markers used.Furthermore the wavelength or wavelength bands λ₁, . . . , λ_(m), andλ₁, . . . , λ_(n) may interleave as shown, or the may be arranged in adifferent way with respect to one another.

Light transmitted through the output waveguides 29, 29′ of the lightdirecting means 19 is passed on to the detection means comprising twogroups of detectors 31, 31′, respectively, each having a plurality ofdetectors; each group of detectors 31, 31′ being mounted on planardetector substrate devices 30, 30′, respectively. The substrate devices30 and 30′ may be butt-coupled to the edge of the substrate 80 asdescribed in more detail in FIG. 3 a.

FIG. 3 a shows a detailed view of the interface of the plurality ofoutput waveguides 29 with the detector substrate 30. The detectorsubstrate 30 and the side face of the substrate 80 in the region wherethe waveguides 29 extend to the edge of the substrate 80 are polished,respectively, and butt-coupled. The substrate 30 may be attached to thesubstrate 80 by means of transparent glue or resin. The detectors 31 maybe top-entry photodiodes that are flip-chip bonded over etched v-groovesin the detector substrate 30, that extend from the edge of the substrate30 that is coupled to the edge of the substrate 80 to a locationunderneath the entry face of each photodiode. For this purpose thedetector substrate 30 may conveniently be made of crystalline siliconwhich can be etched to form v-grooves 35 with a selectivecrystallographic etch, such as described in U.S. Pat. No. 4,945,400. Byusing this processing method the v-groove formed in this manner willautomatically terminate in an inclined end face that can be used toreflect light upwards into the entry face of the photodetector. U.S.Pat. No. 4,945,400 also describes a way of coating the end face with ametal layer 32 which extends over the end of the v-groove 35 andsimultaneously serves to enhance the reflectivity of the end face of thegroove and provides for a soldering pad and top contact for thephotodetectors 31. With this arrangement in place signal leads 33, 34can conveniently be attached to the metal layer 32 and the bottomcontact of the photodiode 31 which lead to an electronic processingcircuitry 100.

In an alternative embodiment of the present invention (not shown), thepluralities of detectors 31 and 31′ may be hybridised directly on thesame substrate device 80 that comprises the pluralities of outputwaveguides 29, 29′, respectively. This can be carried out by using thedetector hybridisation technology described in WO9835253 or using othertechniques well known in the art of integrated optics. For example, if asilica or silica on silicon substrate is used for providing thewaveguides, a rectangular recess can be etched into the substrate and atop entry photodiode may be glued down sideways into the recess,abutting the relevant waveguide with its top entry facet. Connections tothe photodiode can then be made by soldering directly to the top andbottom facets of the diode on the portion of the diode protruding fromthe substrate 80.

Another preferred embodiment of the present invention is shown in FIG. 2b. Here, the analyte handling means analyte means 78 is integrated onthe same substrate 20 as the light directing means 19 of the opticaldetection device, which comprises the dispersive elements 28, 28′, theplurality of output waveguides 29, 29′. The detector substrates 30, 30′carrying the pluralities of detectors 31, 31′ are coupled to thesubstrate 20 in the same way as described before for substrate 80 inFIG. 2 a. In this case a separate fibre optic connection between theanalyte means 78 and the light directing means 19 is not necessary.

In yet another embodiment of the invention (not shown) the lightsupplying means 45 may be integrated on the same substrate 20 as well.Whether all or some of the light supplying means, the analyte handlingmeans and the light directing and detection means will be integrated onthe same substrate or be provided on separate substrates which areconnected by optical fibres will depend on the final manufacturingprocesses chosen, the expected manufacturing yields of the final devicesand the specific applications envisaged.

If integration of the light supplying means, the analyte handling meansand the light directing and detection means on separate substrates ischosen, this will allow these components to be exchanged or maintainedseparately, should one of them fail in operation. Further, such anembodiment would have the advantage that these devices can be physicallyseparated by an appreciable distance. In such a situation the low lossof optical communication fibres will allow the separation of the lightsupplying means, the analyte handling means and the light directingmeans by several meters, or even several tens of meters. This would havethe advantage that the analyte handling means can for example be locatedin a higher containment level compartment than the light supplying meansand the light detecting means, or that these devices can be distributedover different locations in the same building.

In a preferred embodiment shown in FIG. 3 b the waveguide 21 splits viaa y-junction into waveguides 23 and 24, which both interface with theanalyte channel 70 at an angle such that light refracted across theboundary of the waveguide with the analyte channel enters the liquidsuspension or solution guided in the channel at an angle of between 30and 60 degrees and more preferably at an angle of 45 degrees within twodifferent interrogation regions 18′ and 18″. On the opposite side of theanalyte channel 70 waveguide 27 collects forward scattered light andwaveguide 26 collect side scattered and fluorescent light emitted fromthe interrogation region 18′ and the waveguide 27′ collects forwardscattered light and waveguide 26′ collects side scattered andfluorescent light emitted from the interrogation region 18″,respectively. The choice of the interface angles of the variouswaveguides and the general design of these interfaces follow the sameprinciples as those described in the previous example of FIG. 2 a. Theadvantage of such an arrangement is that a measurement of the analytecontent is taken at two successive interrogation points 18′ and 18″which allows for a direct determination of the flow speed and dynamicsof the analytes propagating in the analyte channel 70. This may have theadvantage of allowing more accurate sorting of analytes if such wererequired and detecting any differential motion in the analytes

In FIG. 3 b there are also shown absorbing regions 25 to lower the noiselevel of scattered, unguided light travelling around the substrate 20. Aplurality of such doped absorbing regions 25 are included on thesubstrate 20 in the vicinity of the waveguides 23, 24, 26, 27 and theirinterfaces with the analyte channel 70. Such absorbing regions are forexample described in WO 9928772.

A detailed view of the analyte handling means 78 comprising an analytesorting means 72 is shown in FIG. 3 c. The analyte sorting region 72 isformed as a cascade of y-junctions branching the analyte channel 70 into2^(N) sorting channels (N being the number of stages in the cascade) ina plurality 72′ that terminate in a plurality of 2^(N) output ports 74.The number of terminal sorting channels 72′ is preferably greater orequal to the number of fluorescent wavelengths used for detecting theproperties of the analytes. As described above, the sorting channels inthe plurality 72′ as well as the analyte channel and their y-junctionsare formed as trenches etched into the substrate 20, e.g. by means of areactive ion etch process, and the ports 70 and 74 are formed asextended recesses in the substrate 20 by means of a similar etchprocess.

Each analyte channel y-junction in the analyte sorting means 72 containsa pair of sorting elements 73. In the preferred embodiment shown, thesorting elements 73 are opposite polarity electrodes that are buried oneither side of the analyte channels in the sorting means 72 adjacent toeach y-junction as extended recesses. FIG. 4 b shows a cross section ofthe analyte channel 70 and the electrodes 73 along the line B—B of FIG.3 c. The electrodes 73 extend on either side of the etched analytechannel into the substrate device 20 from the top of the substrate. Thiscan be achieved by etching the substrate on either side of the analytechannel with a directional etch process, such as a reactive ion etch andsubsequently depositing a metal layer through a mask into the etchedrecesses. As shown in the Figure, the top surface area of each electrode73 is enlarged to allow for convenient connection of electrical leadtraces 75 (shown in FIG. 3 c) extending over the top of the substrate20. The electrodes 73 may be in direct contact with the analyte channel70 or they may be separated from the channel by dielectric walls in thesubstrate, as shown in FIG. 4 b. As is shown in detail in FIG. 3 c, onthe surface of the substrate device 20 a plurality contact traces 75 isformed by lithographic processes that extends from the electrodes 73 tothe edge of the substrate 20 allowing for an electronic control unit 76to be connected to the contact traces 75 via a plurality of externalleads 77 for controlling the electrodes 73 in FIG. 3 c.

When the detection section is used for analyte sorting the analytes usedare either negatively or positively charged. This can be achieved, forexample, by coating all analytes with microbeads via a common surfacemarker, whereby the beads will carry a negative or positive ionic chargein suspension or solution, e.g. due to a coating of the beads with Ni Feor Cu ions. Analytes that pass the interrogation region 18 areilluminated by light emitted at the light supplying means andtransmitted by the waveguide 21. The light emerging from theinterrogation region 18 is collected by the waveguides 26, 27 and isguided to the detectors 31. If the width of the analyte channel, theflow rate of the analytes and the concentration of the analytes are allsuch that only one particle to be analysed passes the waveguide 21 at atime, the speed of the analytes in the channel can be accuratelypredicted. Taking into account the hydrodynamic properties of thesorting region 72 an accurate determination can be made as to when theanalyte particle passes each y-junction and as appropriate each in theplurality of pairs of electrodes 73 can be used to redirect each analyteparticle into a y-junction branch according to the fluorescentwavelength detected. In such a case, the electric field applied to eachin the plurality of pairs of electrodes 73 should be strong enough toredirect the flow of a particle, but not so strong that the particlegets immobilised against the wall of the analyte channel.

As described above the analyte channel 70 and the analyte sortingchannels 72′ are formed as extended recesses in the substrate device 20,which means they are open towards the top surface of the substratedevice 20. To seal the channels a substrate 60 extending over the areaaround the analyte channel 70 and the analyte sorting channels 72′ isbonded to the top surface of substrate 20. The horizontal boundary ofthe substrate 60 corresponds to the dashed lines in FIG. 2 b. Thesubstrate presence 60 is shown in more detail in FIG. 2 c. At thelocation of the analyte input port 71 and of each of the plurality ofoutput ports 74 apertures 61, 62 are formed in the sealing substrate 60such that liquid guiding means 63, 64 can be connected to each port. Across section of the interface between the liquid guiding means 63 withthe aperture 61 in the region of the input port 71 is shown in furtherdetails in FIG. 4 a. Bonding of the substrate device 60 to the substratedevice 20 may be achieved simply by polishing both bond surfaces andcreating a contact bond which is maintained by the van der Waalsinteractions between the substrates 20 and 60. To be able to polish thesurface of the substrate 20, either a buried waveguide structure willhave to be used, or, when if silicon-on-insulator ridge waveguides areused, an additional amorphous silica or other layer may have to bedeposited on top of the waveguide structures and be polished back to asmooth surface before the bonding is carried out. Other means of bondinginclude glues or resins, or soldering, according to methods well knownin the art, but it is important that no such glues, resins or soldersinterfere with the optical properties of the optical waveguides or theanalyte channel on the substrate.

A cross-section of the connection between the substrate device 20 andthe substrate 60 in the region of the analyte input port 71 is shown inFIG. 4 c. Substrate 20 comprises a recess for the liquid input port 71.The aperture 61 in the substrate 60 is located exactly above the port71. On the top surface of substrate 60 there is positioned the liquidinput guiding means 63, which has a flanged connection with thesubstrate 60. The liquid output guiding means 64 are arranged in thesame manner above the output ports 74.

In one embodiment the analyte flow is injected into the input port 71through the liquid guiding means 63, preferably at a predetermined,constant flow rate. The analyte flow is directed through the analytechannel 70 via the sorting means 72 to the output ports 74 where it isreceived by the liquid output guiding means 64. If it is an objective toreduce the possibility of clogging of the analyte channel 70 withaggregated analytes in suspension, such as clumps of cells, anappropriate pre filter can be used.

In still another embodiment of the analyte handling means according toFIG. 5 a, there is provided a first liquid guiding means in the form ofan input channel 200 for introducing a sheath fluid into the analytechannel and a second liquid guiding means in the form of a second inputchannel 210 for introducing the analyte suspension or solution. Thechannel 210 is concentric with the channel 200 in the region above thesubstrate 60. Following the direction of the analyte flow the channel200 then interfaces with the substrate 60, whereas the channel 210extends into the cavity of the input port 71, which is over-etchedvertically to accommodate this additional device. The channel 210 thencurves by 90 degrees towards the analyte channel 70 and ends with aconical nozzle 211 pointing towards the channel 70 for introducing theanalyte suspension or solution into the analyte channel 70. The sheathfluid is introduced in the first input channel 200 with a velocity thatis at least equal to or higher than the velocity of the analytesuspension or solution, emerging from the nozzle 211, such that when theanalyte liquid suspension or solution is introduced out of the nozzle211 into the surrounding flow of sheath fluid, both the analyte flow andthe flow of the sheath fluid will converge into the analyte channel 70and the analyte flow will be focused into the centre of the crosssection of the analyte channel 70 according to the Bernoulli-Effect ofhydrodynamic focusing that was described previously. As a result theanalytes are confined in the centre region of the analyte channel,forming an analyte flow in the centre of a parabolic pressuredistribution along the cross-section of the analyte channel 70.

As is usual in flow cytometry applications, both the sheath fluid andthe buffer fluid for the analyte suspension or solution will bephosphate buffered saline or a similar electrolyte solution.

FIG. 5 b illustrates an arrangement for providing the sheath fluid andthe liquid suspension or solution, for use with the hydrodynamicfocusing assembly shown in FIG. 5 a. For simplicity, the waveguides arenot shown on the substrate 20 in this figure. The sheath fluid is pumpedby a pump 220 from the reservoir 260 through the one of pre-filters 290and channel 200 to the input 71 of the analyte channel analyte channel70. A second pump 230 will pump suspension or solution buffer from thereservoir 270 to the analyte injection valve 280. The pumps 220 and 230may be similar to those commonly used in protein liquid chromatographyand the injection valve 280 may be an injection valve commonly used inliquid chromatography applications. An injection loop 240 is then usedin conjunction with the valve 280 to bring a sample of analytesuspension or solution in line with the analyte sample buffer flowemerging from pump 230 which is conducted via the channel 210 throughthe other of prefilters 290 to the input 71. The pumps 220, 230 and theinjection loop 240 are controlled electronically by a controller 250.

In case of an embodiment of the analyte handling means 78 according toFIG. 3 a, in which two incident waveguides 23, 24 are and four emergentwaveguides 26, 26′, 27, 27′ are used, twice the number of dispersiveelements in the light directing means 19 and groups of detectors 31 inthe detection means may be used to detect the signals collected by thewaveguides 26, 26′, 27, 27′.

In still an other embodiment of the present invention any or allelements, i.e. the light supplying means, the analyte sorting andinterrogation region 18 and the signal detection region may betemperature controlled in order to ensure a uniform and repeatableoperating environment. Due to the substrate integration of both theoptical and fluidic components of the system this can be achievedconveniently by mounting the respective substrates on Peltier coolingdevices. Such devices are available commercially and they will allow fora compact design of a cooled packaged device.

The present invention in its broader aspects is not limited to thespecific details illustrated in the foregoing examples and shown in thefigures. Various modifications may be made without departing from thespirit of the invention.

If, for example, only fluorescent detection is required and not analytesorting, it may favourable that the analyte channel 70 be unbranched,i.e. it would not contain an analyte sorting region 72 and the devicewould only have a single analyte-output port 74.

In another embodiment the analyte channel 70 of the analyte handlingmeans extends along an edge of the substrate device 20 such that lightilluminating the analytes in the interrogation region 18 in the analytechannel can be received directly from the edge of the substrate.

As mentioned before, in certain embodiments of the invention the numberof fluorescent wavelengths may be larger than the number of incidentwavelengths, i.e. some or all of the fluorescers may share a commonwavelength for their absorption maximum while each having a differentwavelength for their emission maximum. Such a method is commonly used inprior art devices, as this will reduce the complexity of the lightsupplying means. On the other hand restricting the number of excitationwavelengths may reduce the number of appropriate fluorescers available,as it may not be possible to find a sufficiently large number offluorescers for any given application that share a common excitationmaximum while having different fluorescent emission maxima. In any eventa device constructed according to the present invention, as describedabove, will provide substantially improved design flexibility in findingthe ideal trade-off between the number of excitation wavelengths usedand the fluorescent dyes or particles chosen for any given application.

In another embodiment of the invention light incident on theinterrogation region 18 may be introduced to interrogation region 18 bydirecting it along the analyte channel 70 instead of guiding it along awaveguide 21 through the substrate 20, i.e. the analyte channel itselfis used as a light waveguide. In this case the incident light is guidedparallel to the liquid flow into the interrogation region 18.

1. Device for analysing analytes in a liquid suspension or solution comprising: analyte handling means including an analyte input region, and an analyte channel for carrying analytes in a liquid suspension or solution through a light receiving region to an analyte output region; light supplying means for supplying light to the light receiving region to illuminate an interrogation region of the analyte channel, the light supplying means comprising a first input optical waveguide for directing light into a first interrogation region; and light guiding means for directing light emerging from the analytes in the interrogation region to optical detection means for detecting one or more properties of the analytes in the suspension or solution, the light guiding means comprising, a first output optical waveguide positioned to receive forward scattered light emerging from the first interrogation region, and a second output optical waveguide positioned to receive side scattered light emerging from the first interrogation region at an angle to the forward scattered light, at least the analyte channel, and the first and second output optical waveguides being integrated on the same first planar substrate.
 2. Device as claimed in claim 1, wherein the first input optical waveguide interfaces with the analyte channel such that it directs light into the analyte channel at an angle of between 30 and 60 degrees from the longitudinal axis of the analyte channel.
 3. Device as claimed in claim 2, wherein the first output optical waveguide interfaces with the analyte channel, such that it collects light emerging from the analyte channel at an angle of between 30 and 60 degrees from the longitudinal axis of the analyte channel.
 4. Device as claimed in claim 3, wherein the second output optical waveguide is interfacing with the analyte channel, such that it collects light emerging from the analyte channel at an angle of between 60 to 120 degrees from the light collected by the first output optical waveguide.
 5. Device as claimed in claim 1, wherein the light supplying means comprises a second input waveguide for directing light into a second interrogation region in the analyte channel spaced from the first interrogation region and the light guiding means comprises a third output optical waveguide positioned to receive forward scattered light from the second interrogation region and a fourth output optical waveguide positioned to receive side scattered light emerging from the second interrogation region at an angle to the forward scattered light, at least the third and fourth output optical waveguides also being integrated on the first planar substrate.
 6. Device as claimed in claim 1, wherein doped absorbing regions are provided in the first planar substrate adjacent to the analyte channel or the optical waveguides for reducing the amount of unguided light propagating in the substrate.
 7. Device as claimed in claim 1, wherein the refractive index of any or all of the optical waveguides substantially matches the refractive index of the liquid suspension or solution.
 8. Device as claimed in claim 1, wherein one or more of said optical waveguides is tapered at its interface with the analyte channel.
 9. Device as claimed in claim 1, wherein one or more of said optical waveguides is provided with a single or multiple dielectric coating at its interface with the analyte channel.
 10. Device as claimed in claim 1, wherein the light supplying means comprises a light source for emitting light with a predetermined characteristic, which is changeable by interaction of the light with the analytes in the suspension or solution according to the properties of the analytes.
 11. Device as claimed in claim 1, wherein the light supplying means comprises a light source for emitting light of one or more discrete wavelengths or wavelength bands λ, λ₁. . . λ_(m).
 12. Device as claimed in claim 11, wherein the one or more wavelengths or wavelength bands λ, λ₁. . . λ_(m) is selected according to the properties of the analytes to be investigated.
 13. Device as claimed in claim 11, wherein the optical detection means can discriminate the one or more wavelengths or wavelength bands λ, λ₁. . . λ_(m) and one or more wavelength or wavelength bands λ′₁. . . λ′_(n) emitted by the analytes as a consequence of fluorescence.
 14. Device as claimed in claim 13, wherein none of the wavelength bands λ₁. . . λ_(m), λ′₁. . . λ′_(n) have an overlap with one another of more than −20 dB.
 15. Device as claimed in claim 14, wherein none of the wavelength bands λ₁. . . λ_(m), λ′₁. . . λ′_(n) have an overlap of more than −30 dB.
 16. Device as claimed in claim 1, wherein first liquid guiding means is provided for introducing sheath fluid into the analyte channel and second liquid guiding means is provided for introducing the analyte suspension into the analyte channel, wherein the first and second liquid guiding means are formed such that they allow for hydrodynamic focusing of the anlayte suspension or solution into the centre of the analyte channel.
 17. Device as claimed in claim 1, wherein the analyte channel is machined or etched into a top surface of the first planar substrate and the analyte channel is open towards the said top surface, and wherein the analyte channel is sealed by attaching a further substrate to the first substrate that extends over the area of the analyte channel.
 18. Device as claimed in claim 17, wherein apertures are provided in the further substrate, such that said liquid guiding means can be connected to the analyte channel for injecting liquid into and receiving liquid from the analyte channel via said apertures.
 19. Device as claimed in claim 1, wherein the analyte channel extends along an edge of the first substrate such that light illuminating the analytes in the first interrogation region can be received from the edge of the substrate.
 20. Device as claimed in claim 1, wherein the first input waveguide and the analyte channel are formed by the same recess in the first substrate, such that incident light is introduced into the first interrogation region by directing light along the analyte channel.
 21. Device as claimed in claim 1, comprising analyte sorting means for sorting analytes in a liquid suspension or solution received from the analyte output region, comprising a plurality of sorting channels comprising at least one y-junction integrated on the first planar substrate, wherein a pair of opposite polarity electrodes are formed in the first planar substrate on either side each of said y-junctions.
 22. Device as claimed in claim 21, wherein the plurality of sorting channels is formed as a cascade of p-junctions comprising at least two stages.
 23. Device as claimed in claim 21, wherein a plurality of contact traces are formed on the first planar substrate for connecting an external computer controlled power supply to each pair of opposite polarity electrodes formed in the first planar substrate.
 24. Device as claimed in claim 1, wherein the light supplying means comprises one or more components integrated on a second planar substrate optically connected to the first planar substrate.
 25. Device as claimed in claim 24, wherein one or more light emitting diodes or laser diodes are provided on the second planar substrate.
 26. Device as claimed in claim 25, wherein at least one integrated optical waveguide for carrying light from each of the light emitting diodes or laser diodes is provided on the second planar substrate.
 27. Device as claimed in claim 26, wherein a dispersive element is integrated on the second planar substrate for combining light of different wavelengths λ₁. . . λ_(m), received from a plurality of waveguides into a single waveguide.
 28. Device as claimed in claim 27, wherein the dispersive element is an arrayed waveguide grating or a transmission grating integrated on the second planar substrate.
 29. Device as claimed in claim 13, wherein the light guiding means comprises one or more components integrated on a third planar substrate optically connected to the first planar substrate.
 30. Device as claimed in claim 29, wherein at least one dispersive element, at least one optical waveguide for carrying light towards each dispersive element, and a plurality of output waveguides for collecting light of wavelengths or wavelength bands λ₁. . . λ_(m), λ′₁. . . λ′_(n) output from each dispersive element are integrated on the third planar substrate.
 31. Device as claimed in claim 30, wherein the dispersive element is an arrayed waveguide grating or a transmission grating integrated on the third planar substrate.
 32. Device as claimed in claim 30, wherein a dispersive element, an optical waveguide for carrying light towards the dispersive element, and a plurality of output waveguides for collecting light of wavelengths or wavelength bands λ₁. . . λ_(m), λ′₁. . . λ′_(n) output from the dispersive element are provided (1) for separating forward scattered light received from the first output optical waveguide and (2) for separating side scattered light received from the second output optical waveguide.
 33. Device as claimed in claim 29, wherein the light guiding means comprises at least one optical detector integrated in or hybridised on a fourth planar substrate.
 34. Device as claimed in claim 33, wherein a plurality of optical detectors are provided on the fourth planar substrate with one optical detector being provided for detecting each of a plurality of wavelengths λ₁. . . λ_(m), λ′₁. . . λ′_(n).
 35. Device as claimed in claim 34, wherein one or more of the optical detectors are photodiodes.
 36. Device as claimed claim 35, wherein each optical detector is mounted over an inclined end face at the end of a v-groove formed in the fourth substrate, the v-groove extending to the edge of the substrate.
 37. Device as claimed in claim 33, wherein the fourth substrate is butt-coupled to the third substrate and bonded thereto by means of glue or resin.
 38. Method for investigating the properties of one or more analytes in a liquid suspension or solution, comprising: carrying analytes in a liquid suspension or solution in an analyte channel through a light receiving region; supplying light to the light receiving region to illuminate an interrogation region of the analyte channel, wherein said supplying light supplies light using a first input optical waveguide into a first interrogation region; directing light emerging from the analytes in the interrogation region to at least one optical detector configured to detect one or more properties of the analytes in the suspension or solution, wherein said directing light comprises, receiving forward scattered light emerging from the first interrogation region in a first output optical waveguide, and receiving side scattered light emerging from the first interrogation region at an angle to the forward scattered light in a second output optical waveguide, wherein at least the analyte channel and the first and second output optical waveguides are integrated on the same first planar substrate; and outputting data representative of said received scattered light to a receiving device or a user.
 39. Method as claimed in claim 38, wherein the optical properties of the one or more analytes in the suspension or solution are marked by markers with known optical properties, and wherein the supplying light further comprises providing one or more wavelength or wavelength bands λ₁, . . . , λ_(m), according to emission and absorption characteristics of the used markers.
 40. Method as claimed in claim 39, wherein the markers for the analytes are fluorescent.
 41. Method as claimed in claim 38, wherein at least two wavelengths or wavelength bands λ₁. . . λ_(m), λ′₁. . . λ′_(n) in the light emerging from the first interrogation region are detected simultaneously.
 42. Method as claimed in claim 38, wherein the flow of the analyte suspension or solution through the analyte channel or sorting channels is reversible.
 43. Method as claimed in claim 38, wherein a sheath fluid introduced by a first input channel confines the analyte suspension or solution introduced by a second input channel in the centre of the analyte channel.
 44. Method as claimed in claim 38, wherein the light illuminating the analytes in the first interrogation region is introduced to the first interrogation region by directing the light along the analyte channel.
 45. Method for investigating the properties of one or more analytes in a liquid suspension or solution, comprising: carrying analytes in a liquid suspension or solution in an analyte channel through a light receiving region; supplying light to the light receiving region to illuminate an interrogation region of the analyte channel, wherein said supplying light supplies light using a first input optical waveguide into a first interrogation region; directing light emerging from the analytes in the interrogation region to at least one optical detector configured to detect one or more properties of the analytes in the suspension or solution, wherein said directing light comprises, receiving forward scattered light emerging from the first interrogation region in a first output optical waveguide, and receiving side scattered light emerging from the first interrogation region at an angle to the forward scattered light in a second output optical waveguide, wherein at least the analyte channel and the first and second output optical waveguides are integrated on the same first planar substrate; and storing data representative of said received scattered light. 